The present invention relates to new strains of the Modified Vaccinia virus Ankara (MVA) that have a strongly reduced virulence for most mammals, especially humans, but nevertheless grows in cells of a continuous cell line approved for the production of a therapeutic agent such as a vaccine. The invention also relates to a method for producing said adapted MVA strains. The MVA can be used e.g. for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system.
An organism is constantly challenged by infectious agents like bacteria, viruses, fungi or parasites. The immune system prevents the organism from permanent infection caused by these agents by the destruction and elimination of these infectious agents and any toxic molecules produced by them. The immune system can be divided into a specific and an unspecific part although both parts are closely cross linked. The unspecific immune response enables an immediate defense against a wide variety of foreign substances and infectious agents. In contrast, the specific immune response is raised after a lag phase, when the organism is challenged with a substance for the first time. However, the specific immune response is highly efficient. The specific immune response is responsible for the fact that an individual who recovers from a specific infection is protected against this specific infection but still susceptible for other infectious diseases. In general, a second infection with the same or a very similar infectious agent causes much milder symptoms or no symptoms at all. The immunity persists for a long time, in some cases even lifelong. This immunological memory is used for vaccination, where the organism is challenged with a harmless or inactivated form of the infectious agent to induce a specific immunity. Sometimes adjuvants are incorporated into vaccines to enhance the specific immune response.
Much of the knowledge about infectious diseases and immunity is contributed by studies of smallpox. The disease is caused by the variola virus, a member of the genus of Orthopox viruses. Nearly two centuries ago, prophylactic inoculations with cowpox was initiated resulting in the immunization against smallpox. Later immunization was performed with the Vaccinia virus. In the early 1950s, many of the industrialized countries had eliminated endemic smallpox by using vaccination with Vaccinia virus. However, smallpox vaccination with this Vaccinia virus resulted occasionally in serious complications, such as postvaccinal encephalitis, generalized Vaccinia or contact infection.
A new vaccine that does not show these complications was developed by Anton Mayr. The pox vaccine consists of the pox virus Modified Vaccinia Virus Ankara (MVA) and was used for parenteral vaccination against smallpox in about 150 000 vaccinations without causing any complications related to the vaccination. Even children with immunologic deficiencies did not show serious side effects. The MVA was obtained by mutation and selection of the original vaccina virus Ankara after 575 passages in chicken embryo fibroblast cultures. The safety of this MVA is reflected by biological, chemical and physical characteristics. MVA has a reduced molecular weight, six deletions in the genome, and is highly attenuated for mammalian cells, i.e. DNA and protein is synthesized but virtually no viral particles are produced. The Modified Vaccina virus Ankara developed by Anton Mayr was deposited at the European Collection of Cell Cultures (ECACC), Salisbury, UK, under depository No. V 94012707.
The vaccination against smallpox was highly successful. In 1979, the World Health Organization declared the eradication of smallpox. Accordingly, the mass vaccination of children was discontinued and only laboratory workers and members of the armed forces of some countries are vaccinated.
With the eradication of smallpox, the predominant cause of pox infection in humans was removed. However, some non-human poxviruses have reduced host specificity, i.e. they cause infections not only in their typical host (e.g. for cowpox the cow), but also in other animals, (e.g. rats and cats). Humans can be infected by this route as well. Since parts of the population are no longer immune against smallpox, orthopox infections of animal species can be dangerous for them. Domestic animals are the main source of infection for humans. Accordingly, the vaccination of domestic animals against orthopoxviruses is of increasing importance. In addition, the MVA may be of significance as a vector for gene therapy, i.e. to transfer nucleic acid sequences into a target cell where they are expressed.
For a logarithmic reproduction of the MVA cell cultures of primary or secondary chicken embryo fibroblasts are needed. The cells are obtained from chicken eggs that are incubated for 10 to 12 days. Since eggs are subjected to a biological variability, the cells obtained for the cell culture system are variable on a cellular level as well. In addition, in a chicken embryo xe2x80x9cfibroblast culturexe2x80x9d often other cell types such as epithelial cells are found. This variation of the cells also results in variation of viruses produced in chicken embryo fibroblasts. It is therefore difficult to standardize and validate the cell culture system to guarantee a constantly high quality of the MVA produced. Furthermore, contamination of the cell culture system by microorganisms or viruses already present in the incubated eggs can not be completely excluded. When the MVA is grown in virus-contaminated cells, the MVA may recombine with the contaminating virus. Thereby an MVA with new and unpredictable characteristics may be generated. For the production of the virus in large scale in a suspension culture, primary or secondary chicken embryo fibroblasts are also not highly suitable. In addition, the purification and concentration of MVA by ultra gradient centrifugation would be advantageous. However, such purification is difficult, when MVA is cultivated on primary or secondary chicken embryo fibroblast. Finally, an increasing number of patients have developed allergies against chicken egg""s albumen. Although the in vitro conditions of the cultivation strongly reduce the allergenic potential, a hazard of an allergic reaction can not be completely excluded.
In conclusion, on the one hand the MVA can only be efficiently grown in primary or secondary chicken embryo fibroblasts causing a number of disadvantages, however, on the other hand the save application of the MVA in humans has been shown by its large-scale application as a vaccine.
It is an object of the present invention to provide conditions for the production of homogeneous virus particles of the MVA. A Additionally, said conditions should allow an easy and large-scale production of the MVA.
To achieve the foregoing and other objects, the present invention provides an MVA strain that is adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic agent.
According to the present invention, for the first time an efficient and large-scale production of MVA is possible. Since cells of a continuous cell line are homogeneous and their characteristics are stable the MVA harvested from these cell lines is also homogeneous with highly predictable characteristics. Furthermore, the risk of contamination by microorganisms can be controlled and contamination of the MVA preparation by proteins of the chicken eggxe2x80x94as found when cultivating MVA on chicken embryo fibroblastsxe2x80x94can be excluded. The handling of a permanent cell line is convenient and thus highly suitable for industrial application.
In a preferred embodiment of the invention, the MVA is adapted for growing in cells of a mammalian cell line, which is approved for the production of a vaccine. It has been surprisingly found that the MVA adapted to a mammalian cell line such as the Vero cell line still has a reduced virulence for humans and also for a wide range of other mammals. Accordingly, the MVA is highly attenuated i.e. DNA and protein is synthesized but virtually no viral particles are produced, resulting in a virtually eliminated disease-causing capacity. Hence, the MVA according to the present invention is also highly suitable as a vaccine for humans and for a wide range of mammals. Accordingly, the MVA is especially applicable in the veterinary field.
Furthermore, a method to obtain an MVA strain according to the present invention is provided. According to this embodiment of the invention, cells of a cell line that is approved for the production of a therapeutic substance, are infected with the wild-type MVA. Preferably a high multiplicity of infection (MOI), i.e. a high number of viruses per cell is used for this infection. Then, the viruses are harvested and fresh cells of the same cell line are infected with the newly produced viruses. Said process is repeated (serial passaging) until the MVA is adapted to said cell line. Adaptation is reached, when 72 h post infection, the virus titer is at least 1- to 9-fold, preferably 10- to 99-fold, more preferably 100- to 106-fold, and most preferably more than 107- to 1010-fold increased compared to the input virus titer. The adaptation is reached after a limited number of passages.
xe2x80x9cAdapted for growingxe2x80x9d means that the amount of virus produced from an infection (Output) is increased compared to the amount of virus originally used to infect the cells (Input). In this case the Output/Input ratio is greater than 1.
xe2x80x9cDerivativexe2x80x9d of the MVA deposited at ECACC, Salisbury, UK, under the depository number 99101431 and/or provisional accession number 01021411 means an MVA which is adapted for growing in Vero cells at a rate, which is essentially the same as the growth rate of the deposited strain but carriers at least one difference in its genome compared to the deposited strain.
The term xe2x80x9cimmune systemxe2x80x9d basically describes a complex involved in the defense of the organism against foreign substances and microorganisms. It is divided into a cellular part comprising several cell types, such as e.g. lymphocytes and other cells derived from white blood cells, and a humoral part comprising peptides and proteins such as antibodies, complement factors, and cytokins.
The term xe2x80x9cimmune responsexe2x80x9d describes the reaction of the immune system, when a foreign substance or microorganism enters the organism. Generally, the immune response is divided into a specific and an unspecific reaction although both are closely cross linked. The unspecific immune response is regarded as the immediate defense against a wide variety of foreign substances and infectious agents. The specific immune response can be characterised as a highly efficient defense mechanism of the organism against a foreign substance which is raised against said substance after a lag phase and highly specific for said substance. The specific immune response is responsible for the phenomenon that an individual who has recovered from a specific infection is protected against this specific infection in future.
xe2x80x9cActivator of the immune systemxe2x80x9d means any substance capable of provoking or enhancing an immune response.
xe2x80x9cSuppressor of the immune systemxe2x80x9d means any substance capable of reducing or inhibiting an immune response.
xe2x80x9cStabilizer of the immune systemxe2x80x9d means any substance capable of keeping the immune response on a constant level.
The inventors provide two preferred MVA strains that are adapted to an African green monkey cell line, called Vero cell line (ATCC No. CCL-81). The MVA-strain, which was passaged 100-times in Vero cells was called xe2x80x9cVero-MVAxe2x80x9d and deposited on Oct. 14, 1999, under the Budapest Treaty of 1977 at the European Collection of Cell Cultures Salisbury, UK under depositary No. 99101431. The MVA strain after 200 passages in Vero cells was called xe2x80x9cVero-MVA-200xe2x80x9d and deposited on 14, Feb. 2001, under the Budapest Treaty of 1977 at ECACC under provisional accession number 01021411.
The MVA obtained as described above is further amplified by cultivating the cells of the approved cell line under suitable conditions, infecting cells with the MVA and harvesting the viral particles produced by said cells. Hence the MVA can efficiently and easily be amplified in large-scale. Surprisingly, the MVA of the invention does not increased virulence in cells other than Vero cells such as human cell lines including HL, HEP-2 or HeLA.
In another embodiment of the invention, the MVA contains at least one heterologous nucleic acid sequence i.e. a nucleic acid sequence that is not naturally found in the MVA genome (recombinant MVA). Preferably, the heterologous nucleic acid sequence is a gene, more preferably a gene encoding an immunizing protein, and most preferably encoding a protein immunizing against malaria, rabies and/or hepatitis. The expression of said heterologous nucleic acid sequence is preferably under the transciptional control of a vaccinia virus promoter, more preferably of an MVA-own promoter. In a further preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at a naturally occurring deletion site in the MVA genome (disclosed in PCT/EP96/02926).
The recombinant MVA is used for the introduction of a nucleic acid sequence into a target cell, said nucleic acid sequence being homologous or heterologous to the target cell. The introduction of a heterologous nucleic acid sequence into a target cell may be used to produce heterologous nucleic acids, peptides and/or polypeptides and/or proteins encoded by said nucleic acid sequence in vitro. This method comprises the infection of a host cell with the recombinant MVA cultivation of the infected host cell under suitable conditions, and optionally isolation and/or enrichment of the peptide and/or protein produced by said host cell.
Furthermore, the introduction of a homologous or of a heterologous sequence may be applied for in vitro and preferably in vivo gene therapy. For in vitro and ex vivo gene therapy respectively, cells are isolated from the individual to be treated, transformed with the recombinant MVA and reintroduced into the individual the cells were taken from. For in vivo gene therapy, the recombinant MVA is directly administered to the living animal body including the human body. In a preferred embodiment of the invention, the recombinant MA expresses an antigen or an antigenic epitope. Most preferably, said vector expresses an antigenic determinant from Plasmodium falciparum, Mycobacteria, Herpes virus, Influenza virus, hepatitis, or a human immunodeficiency virus.
Since the MVA according to the invention isxe2x80x94surprisinglyxe2x80x94still highly attenuated, the MVA is ideal to immunize a wide range of mammals including humans. Hence, the present invention also provides a vaccine comprising the MVA for the immunization of a living animal body including a human against pox infections, preferably orthopox infections. The vaccine may contain in addition to the MVA one or more additives such as an antibiotic, a preservative, or a stabilizer. The vaccine is especially applicable in the veterinary field, e.g. for the immunization of animals against orthopox infections such as cats against cat pox, mice against ectromelia or camels against camelpox. The immunization is preferably performed parenterally.
The immunizing effect of an antigenic determinant in a vaccine is often enhanced by the addition of a so-called adjuvant. An adjuvant co-stimulates the immune system in an unspecific manner causing a stronger specific immune reaction against the antigenic determinant of the vaccine. According to another embodiment of the invention, the MVA is used as an adjuvant, to co-stimulate the immune response against the antigenic determinant of a vaccine. In this case it is preferred that the MVA is inactivated. The inactivation of the MVA may be performed e.g. by heat or chemicals. Preferably, the MVA is inactivated by xcex2-propiolacton. According to this embodiment of the invention, the inactivated MVA may be added to vaccines against numerous infectious diseases to increase the immunity against this disease.
In case of an infection, the immune, the nervous, the hormonal and the vascular system of an individual work closely together. These interactions can be regulated by elements of the unspecific immune system e.g. cytokines such as interferons and interleukins. Pox viruses can influence the regulation of the immune system (Swiss Vet 11/99, 13-17). Hence, in a further embodiment of the invention, the MVA and preferably the inactivated MVA is used in mammals including humans to regulate the cellular and humoral elements of the unspecific (innate) immune system. Preferably the MVA is used as a bioregulator, wherein dysfunctions of the immune system are eliminated and the body""s own defense mechanisms are activated, stabilized and/or suppressed. Most preferably, the MVA is used as a bioregulator in case of a viral infection e.g. with herpes, hepatitis B or C virus, in case of a chronic inflammatory disease and/or to support tumor therapy. The MVA may also be used to stabilize the immune system in a situation of increased susceptibility against infections such as in the case of stress or in neonatals. The active and/or preferably the inactivated MVA can be applied systemically e.g. intramuscularly and/or locally e.g. through mucous membranes and/or skin.
In conclusion, the present invention provides MVA strains that can in general be used for the same applications as the wild-type MA, but eliminate the problems caused by the amplification of the wild-type MVA in chicken embryo fibroblasts.
The invention inter alia comprises the following, alone or in combination:
A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic substance.
The MVA as above adapted for growing in cells of a mammalian cell line.
The MVA as above, wherein the cell line is approved for the production of a vaccine.
The MVA as above, wherein said approved cell line is a Vero cell line.
The MVA as above, wherein said approved cell line is the Vero cell line ATCC No. CCL-81.
The MVA as above, deposited at the European Collection of Cell Cultures, Salisbury, UK under depositary No. 99101431 and/or a derivative thereof.
The MVA as above, deposited at the ECACC, Salisbury, UK, under provisional accession number 01021411 and/or a derivative thereof.
The MVA as above, comprising at least one heterologous nucleic acid sequence.
The MVA as above comprising a heterologous nucleic acid sequence coding e.g. for a therapeutic protein and/or an antigenic determinant such as a peptide immunizing against malaria, hepatitis and/or rabies infection.
A host cell infected by the above described MVA.
A composition, preferably a pharmaceutical composition, comprising the. above described MVA and/or the DNA of the MVA.
The pharmaceutical composition described above, wherein the pharmaceutical composition is a vaccine.
The vaccine described above for the immunization of a living animal body including a human.
The vaccine as above for the immunization against an Orthopox infection.
The vaccine as above for the immunization of cats against a cat pox infection, mice against ectromelia infection and/or camels against camelpox infection.
The pharmaceutical composition described above, wherein the MVA is an activator, suppressor and/or stabilizer of the unspecific immune system.
A pharmaceutical composition comprising the above described MVA and/or the DNA of the MVA as an adjuvant.
A pharmaceutical composition comprising the above described recombinant MVA and/or the DNA of the recombinant MVA.
The pharmaceutical composition as described above for use in gene therapy.
A method for introducing a homologous and/or heterologous nucleic acid sequence into a target cell comprising infection of the target cell with the above described MVA.
A method for obtaining an WA strain as described above, comprising a) infection of cells of an approved cell line with a wild-type MVA preferably the MVA deposited at ECACC under depository No. V 94012707, b) harvesting of the viruses, c) infection of fresh cells of the same cell line with the newly produced viruses, and, optionally, d) repetition of b) and c) until the virus is adapted to growth in cells of said cell line.
A method for producing viral particles of the above described MVA, comprising cultivating the cells of an approved cell line under suitable conditions, infecting said cell line with said WA, and harvesting the viral particles produced by said cells.
The method as described above, wherein said cell line is infected with the MVA deposited at the ECACC under depositary No. 99101431 and/or the MVA deposited at the ECACC under provisional accession number 01021411 or a derivative of one of those strains.
A method for producing a nucleic acid sequence, a peptide polypeptide and/or protein, comprising infection of a host cell with the above described recombinant MVA, cultivation of the infected host cell under suitable conditions, and, optionally, isolation and/or enrichment of the nucleic acid sequence, peptide and/or protein produced by said host cell.
Use of the above described WA for producing a pharmaceutical composition for the treatment or prevention of a disease or disorder responsive to said MVA.
Use of the above described MVA for producing a vaccine for the immunization of a living animal body including a human.
Use of the above described WA for producing an activator, suppressor and/or stabilizer of the unspecific immune system.
The use as described above for the manufacture of an adjuvant.
Use of the above described MVA as a vaccine.
Use of the above described MVA as an adjuvant.
Use of the above described MVA as an activator, suppressor and/or stabilizer of the unspecific immune system.
A method for immunization of a living animal body including a human said method comprising administering to a person in need thereof a therapeutically effective amount of an above described pharmaceutical composition.
A method for introducing a homologous and/or heterologous nucleic acid sequence into a target cell comprising infecting the target cell with the above described MVA and/or the DNA of the MVA.
A method for the activation, suppression and/or stabilization of the immune system of a living animal body including a human said method comprising administration of the above described pharmaceutical composition to a living animal body including a human.
A method for enhancing a specific immune response against an antigenic determinant in a vaccine comprising administration of the above described MVA as an adjuvant to a living animal body including a human. A Modified Vaccinia virus Ankara adapted for growing in cells of a continuous cell line obtainable by a process comprising the following steps: infecting cells of a cell line being approved for the production of a therapeutic substance, harvesting the viral particles produced by said cell lines and optionally, repeating the above steps until the desired growth characteristics of said MVA are obtained in said cells.